Project Summary In the proposed studies we seek to disentangle the complex effects of intracellular calcium in the regulation of airway smooth muscle (ASM) contraction. Although biochemical events mediating the effects of calcium on cross-bridge cycling and contraction have been understood and accepted for years, what remains mystifying is how the ASM cell compartmentalizes calcium to regulate the contractile machinery. Our recent findings demonstrate that conventional and simplistic assessments of intracellular calcium signaling have no predictive ability regarding regulation of smooth muscle contractile state. In recent publications we demonstrate the seemingly paradoxical ability of elevated intracellular calcium to mediate relaxation of ASM when stimulated by agonists of bitter taste receptors (TAS2Rs) whereas almost all other G protein-coupled receptors (GPCRs) capable of inducing increased intracellular calcium mediate contraction. Similarly, ligands such as natural flavonoids, chloride channel antagonists, GABAA agonists cause acute ASM relaxation despite causing an acute transient [Ca2+]i increase. Clearly, the disparate effects by GPCRs on ASM contractile state reflect the long-appreciated but poorly-understood ability of the cell to compartmentalize calcium signaling. The relaxant effect of calcium signaling, if understood, could be exploited therapeutically for the management of the airway hyperresponsiveness. To achieve our goal we will combine expertise and strategies in 5 major areas: GPCR biology, biophysics of calcium, cell imaging, proteomics and mathematical modeling. Aim 1 studies will define the mechanisms involved in spatial and temporal distribution of calcium signaling in ASM induced by different GPCR agonists. We will define and localize all proteins, structural elements, and enzymatic activities that regulate calcium distribution in ASM cells stimulated with different GPCR agonists. We will test how disruption of these regulatory elements alters the features of intracellular calcium and the associated regulation of ASM tone. In aim 2 studies, we will employ hyperspectral imaging and phosphoproteomics tools to discern GPCR- specific activation of effector proteins in ASM. With these data we will develop descriptive and predictive mathematical models (Aim 3) that define the key regulatory features enabling compartmentalized calcium signaling and predict the functional consequences of such signaling. The computational models will be validated using experimental data obtained through studies using primary human airway smooth muscle cells and tissues. Our success banks on the unique ability of our team to creatively apply and coordinate cutting edge imaging approaches, multi-fluorophore hyperspectral image analysis, and modeling to well-defined experimental systems that demonstrate disparate and unexplained functional consequences of intracellular calcium. The findings will not only advance the basic science fields of receptor biology and calcium signaling, but also identify targets whose manipulation could be exploited for developing novel bronchodilator therapies.